X-Message-Number: 1390
Date: 03 Dec 92 06:54:37 EST
From: Paul Wakfer <>
Subject: CRYONICS: Freezing Damage (Darwin) Part 2
Perfusion
Perfusion of both groups of animals was begun by carrying out a
total body washout (TBW) with the base perfusate in the absence of any
cryoprotective agent. In the FGP group washout was achieved within 2
- 3 minutes of the start of open circuit asanguineous perfusion at a
flow rate of 160 to 200 cc/min and an average perfusion pressure of 40
mmHg. TBW in the FGP group was considered complete when the
hematocrit was unreadable and the venous effluent was clear. This
typically was achieved after perfusion of 500 cc of perfusate.
Complete blood washout in the FIGP group was virtually impossible
to achieve (see "Results" below). A decision was made prior to the
start of this study (based on previous clinical experience with
ischemic human cryonic suspension patients) not to allow the arterial
pressure to exceed 60 mmHg for any significant period of time.
Consequently, peak flow rates obtained during both total body washout
and subsequent glycerol perfusion in the FIGP group were in the range
of 50-60 cc/min at a mean arterial pressure of 50 mmHg.
Due to the presence of massive intravascular clotting in the FIGP
animals it was necessary to delay placement of the atrial (venous)
cannula (lest the drainage holes become plugged with clots) until the
large clots present in the right heart and the superior and inferior
vena cava had been expressed through the atriotomy. The chest was
kept relatively clear of fluid/clots by active suction during this
interval. Removal of large clots and reasonable clearing of the
effluent was usually achieved in the FIGP group after 15 minutes of
open circuit asanguineous perfusion, following which the circuit was
closed and the introduction of glycerol was begun.
The arterial pO2 of animals in both the FGP and FIGP groups was
kept between 600 mmHg and 760 mmHg throughout TBW and subsequent
glycerol perfusion. Arterial pH in the FGP animals was between 7.1
and 7.7 and was largely a function of the degree of diligence with
which addition of buffer was pursued. Arterial pH in the FIGP group
was 6.5 to 7.3. Two of the FIGP animals were not subjected to active
buffering during perfusion and as a consequence recovery of pH to more
normal values from the acidosis of ischemia (starting pH for FIGP
animals was typically 6.5 to 6.6) was not as pronounced.
Introduction of glycerol was by constant rate addition of base
perfusate formulation made up with 6M glycerol to a recirculating
reservoir containing 3 liters of glycerol-free base perfusate. The
target terminal tissue glycerol concentration was 3M and the target
time course for introduction was 2 hours. The volume of 6M glycerol
concentrate required to reach a terminal concentration in the
recirculating system (and thus presumably in the animal) was
calculated as follows:
Vp
Mc = --------- Mp
Vc + Vp
where
Mc = Molarity of glycerol in animal and circuit.
Mp = Molarity of glycerol concentrate.
Vc = Volume of circuit and exchangeable volume of animal.*
Vp = Volume of perfusate added.
* Assumes an exchangeable water volume of 60% of the preperfusion
weight of the animal.
Glycerolization of the FGP animals was carried out at 10*C to
12*C. Initial perfusion of FIGP animals was at 4*C to 5*C with
warming (facilitated by TBW with warmer perfusate and removal of
surface ice packs) to 10*-12*C for cryoprotectant introduction. The
lower TBW temperature of the FIGP animals was a consequence of the
animals having been refrigerated on ice for the 24 hours preceding
perfusion.
Following termination of the cryoprotective ramp, the animals
were removed from bypass, the aortic cannula was left in place to
facilitate prompt reperfusion upon rewarming, and the venous cannula
was removed and the right atrium closed. The chest wound was loosely
closed using surgical staples.
Concurrent with closure of the chest wound, a burrhole craniotomy
3 to 5 mm in diameter was made in the right parietal bone of all
animals using a high speed Dremel "hobby" drill. The purpose of the
burrhole was to allow for post-perfusion evaluation of cerebral
volume, assess the degree of blood washout in the ischemic animals and
facilitate rapid expansion of the burrhole on rewarming to allow for
the visual evaluation of post-thaw reperfusion (using dye).
The rectal thermistor probe used to monitor core temperature
during perfusion was replaced by a copper/constantan thermocouple at
the conclusion of perfusion for monitoring of the core temperature
during cooling to -79*C and -196*C.
Cooling to -79*C
Cooling to -79*C was carried out by placing the animals within
two 1 mil polyethylene bags and submerging them in an isopropanol bath
which had been precooled to -10*C. Bath temperature was slowly
reduced to -79*C by the periodic addition of dry ice. A typical
cooling curve obtained in this fashion is shown in Figure 5. Cooling
was at a rate of approximately 4*C per hour.
Cooling to and Storage at -196*C
Following cooling to -79*C, the plastic bags used to protect the
animals from alcohol were removed, the animals were placed inside
nylon bags with draw-string closures and were then positioned atop a
6" high aluminum platform in an MVE TA-60 cryogenic dewar to which 2"-
3" of liquid nitrogen had been added. Over a period of approximately
48 hours the liquid nitrogen level was gradually raised until the
animal was submerged. A typical cooling curve to liquid nitrogen
temperature for animals in this study is shown in Figure 6. Cooling
rates to liquid nitrogen temperature were approximately 2*C per hour.
After cool-down animals were maintained in liquid nitrogen for a
period of 6-8 months until being removed and rewarmed for gross
structural, histological, and ultrastructural evaluation.
Rewarming
The animals in both groups were rewarmed to -2*C to -3*C by
removing them from liquid nitrogen and placing them in a precooled box
insulated on all sides with a 2" thickness of styrofoam and containing
a small quantity of liquid nitrogen. The animals were then allowed to
rewarm to approximately -20*C, at which time they were transferred to
a mechanical refrigerator at a temperature of 8*C. When the core
temperature of the animals had reached -2*C to -3*C the animals were
removed to a bed of crushed ice for post-mortem examination and tissue
collection for light and electron microscopy. A typical rewarming
curve is presented in Figure 7.
Modification of Protocol Due To Tissue Fracturing
After the completion of the first phase of this study (perfusion
and cooling to liquid nitrogen temperature) the authors had the
opportunity to evaluate the gross and histological condition of the
remains of three human cryonic suspension patients who were removed
from cryogenic storage and converted to neuropreservation (thus
allowing for post-mortem dissection of the body, excluding the head)
(10). The results of this study confirmed previous, preliminary, data
indicative of gross fracturing of organs and tissues in animals cooled
to and rewarmed from -196*C. These findings led us to abandon our
plans to reperfuse the animals in this study with oxygenated,
substrate-containing perfusate (to have been followed by fixative
perfusion for histological and ultrastructural evaluation) which was
to be have been undertaken in an attempt to assess post-thaw viability
by evaluation of post-thaw oxygen consumption, glucose uptake, and
tissue-specific enzyme release.
Rewarming and examination of the first animal in the study
confirmed the presence of gross fractures in all organ systems. The
scope and severity of these fractures resulted in disruption of the
circulatory system, thus precluding any attempt at reperfusion as was
originally planned.
Preparation of Tissue Samples For Microscopy
Fixation
Samples of four organs were collected for subsequent histological
and ultrastructural examination: brain, heart, liver and kidney.
Dissection to obtain the tissue samples was begun as soon as the
animals were transferred to crushed ice. The brain was the first
organ removed for sampling. The burrhole created at the start of
perfusion was rapidly extended to a full craniotomy using rongeurs
(Figure 8). The brain was then removed en bloc to a shallow pan
containing iced, modified Karnovsky's fixative containing 25% w/v
glycerol (see Table I for composition) sufficient to cover it.
Slicing of the brain into 5 mm thick sections was carried out with the
brain submerged in fixative in this manner. At the conclusion of
slicing a 1 mm section of tissue was excised from the visual cortex
and fixed in a separate container for electron microscopy. During
final sample preparation for electron microscopy care was taken to
avoid the cut edgdes of the tissue block in preparing the Epon
embedded sections.
The sliced brain was then placed in 350 ml of Karnovsky's
containing 25%w/v glycerol in a special stirring apparatus which is
illustrated in Figure 9. This fixation/deglycerolization apparatus
consisted of two plastic containers nested inside of each other atop a
magnetic stirrer. The inner container was perforated with numerous 3
mm holes and acted to protect the brain slices from the stir bar which
continuously circulated the fixative over the slices. The stirring
reduced the likelihood of delayed or poor fixation due to overlap of
slices or stable zones of tissue water stratification. (The latter
was a very real possibility owing to the high viscosity of the 25%w/v
glycerol-containing Karnovsky's.)
Deglycerolization of Samples
To avoid osmotic shock all tissue samples were initially immersed
in Karnovsky's containing 25%w/v glycerol at room temperature and were
subsequently deglycerolized prior to staining and embedding by
stepwise incubation in Karnovsky's containing decreasing
concentrations of glycerol (see Figure 10 for deglycerolization
protocol).
To prepare tissue sections from heart, liver, and kidney for
microscopy, the organs were first removed en bloc to a beaker
containing an amount of ice-cold fixative containing 25% w/v glycerol
sufficient to cover the organ. The organ was then removed to a room
temperature work surface at where 0.5 mm sections were made with a
Stadie-Riggs microtome. The microtome and blade were pre-wetted with
fixative, and cut sections were irrigated from the microtome chamber
into a beaker containing 200 ml of room-temperature fixative using a
plastic squeeze-type laboratory rinse bottle containing fixative
solution. Sections were deglycerolized using the same procedure
previously detailed for the other slices.
Osmication and Further Processing
At the conclusion of deglycerolization of the specimens all
tissues were separated into two groups; tissues to be evaluated by
light microscopy, and those to be examined with transmission electron
microscopy. Tissues for light microscopy were shipped in glycerol-
free modified Karnovsky's solution to American Histolabs, Inc. in
Rockville, MD for paraffin embedding, sectioning, mounting, and
staining.
Tissues for electron microscopy were transported to the
facilities of the University of California at San Diego in glycerol-
free Karnovsky's at 1* to 2*C for osmication, Epon embedding, and EM
preparation of micrographs by Dr. Paul Farnsworth.
Due to concerns about the osmication and preparation of the
material processed for electron microscopy by Farnsworth, tissues from
the same animals were also submitted for electron microscopy to
Electronucleonics of Silver Spring, Maryland.
***Electronucleonics results are not covered here since another
investigator has yet to provide the necessary information and we do
not have access to the pictures.
III. EFFECTS OF GLYCEROLIZATION
Perfusion of FGP Animals
Blood washout was rapid and complete in the FGP animals and
vascular resistance decreased markedly following blood washout.
Vascular resistance increased steadily as the glycerol concentration
increased, probably as a result of the increasing viscosity of the
perfusate.
Within approximately 5 minutes of the beginning of the
cryoprotective ramp, bilateral ocular flaccidity was noted in the FGP
animals. As the perfusion proceeded, ocular flaccidity progressed
until the eyes had lost approximately 30% to 50% of their volume.
Gross examination of the eyes revealed that initial water loss was
primarily from the aqueous humor, with more significant losses from
the posterior chamber of the eyes apparently not occurring until later
in the course of perfusion. Within 15 minutes of the start of
glycerolization the corneal surface became dimpled and irregular and
the eyes had developed a "caved-in" appearance.
Dehydration was also apparent in the skin and skeletal muscles
and was evidenced by a marked decrease in limb girth, profound
muscular rigidity, cutaneous wrinkling (Figure 11), and a "waxy-
leathery" appearance and texture to both cut skin and skeletal muscle.
Tissue water evaluations conducted on ileum, kidney, liver, lung, and
skeletal muscle confirmed and extended the gross observations.
Preliminary observation suggest that water loss was in the range of
30% to 40% in most tissues. As can be seen in Table III, total body
water losses attributable to dehydration, while typically not as
profound, were still in the range of 18% to 34%. The gross appearance
of the heart suggested a similar degree of dehydration, as evidenced
by modest shrinkage and the development of a "pebbly" surface texture
and a somewhat translucent or "waxy" appearance.
Examination of the cerebral hemispheres through the burr hole
(Figure 12) revealed an estimated 30% to 50% reduction in cerebral
volume, presumably as a result of osmotic dehydration secondary to
glycerolization. The cortices also had the "waxy" amber appearance
previously observed as characteristic of glycerolized brains.
The gross appearance of the kidneys, spleen, mesenteric and
subcutaneous fat, pancreas, and reproductive organs (where present)
were unremarkable. The ileum and mesentery appeared somewhat
dehydrated, but did not exhibit the waxy appearance that was
characteristic of muscle, skin, and brain.
Oxygen consumption (determined by measuring the arterial/venois
difference) throughout perfusion was fairly constant and did not
appear to be significantly impacted by glycerolization, as can be seen
Figure 12.
Perfusion of FIGP Animals
As previously noted, the ischemic animals had far lower flowrates
at the same perfusion pressure as FGP animals and demonstrated
incomplete blood washout. Intravascular clotting was serious a
barrier to adequate perfusion. Post-thaw dissection demonstrated
multiple infarcted areas in virtually all organ systems; areas where
blood washout and glycerolization were incomplete or absent. In
contrast to the even color and texture changes observed in the FGP
animals, the skin of the FIGP animals developed multiple, patchy,
nonperfused areas which were clearly outlined by surrounding,
dehydrated, amber-colored glycerolized areas.
External and internal examination of the brain and spinal cord
revealed surprisingly good blood washout of the central nervous
system. While grossly visible infarcted areas were noted, these were
relatively few and were generally no larger than 2 mm to 3 mm in
diameter. With few exceptions, the pial vessels were free of blood
and appeared empty of gross emboli. One striking difference which was
consistently observed in FIGP animals was a far less profound
reduction in brain volume during glycerolization (Figure 13). This
may have been due to a number of factors: lower flow rates, higher
perfusion pressures, and the increased capillary permeability and
perhaps increased cellular permeability to glycerol.
Whereas edema was virtually never a problem during
glycerolization of FGP animals, edema was universal in the FIGP
animals after as little as 30 minutes of perfusion. In the central
nervous system this edema was evidenced by a "rebound" from initial
cerebral shrinkage to frank cerebral edema, with the cortices,
restrained by the dura, often abutting or slightly projecting into the
burrhole. Marked edema of the nictating membranes, the lung, the
intestines, and the pancreas was also a uniform finding at the
conclusion of cryoprotective perfusion. The development of edema in
the central nervous system sometimes closely paralleled the beginning
of "rebound" of ocular volume and the development of ocular turgor and
frank ocular edema.
In contrast to the relatively good blood washout observed in the
brain, the kidneys of FIGP animals had a very dark and mottled
appearance. While some areas (an estimated 20% of the cortical
surface) appeared to be blood-free, most of the organ remained blood-
filled throughout perfusion. Smears of vascular fluid made from renal
biopsies which were collected at the conclusion of perfusion (for
tissue water determinations) revealed the presence of many free and
irregularly clumped groups of crenated and normal-appearing red cells,
further evidence of the incompleteness of blood washout. Microscopic
examination of recirculating perfusate revealed some free, and a few
clumped red cells. However, the concentration was low, and the
perfusate microhematocrit was unreadable at the termination of
perfusion (i.e., less than 1%).
The liver of FIGP animals appeared uniformly blood-filled
throughout perfusion, and did not exhibit even the partial blood
washout evidenced by the kidneys. However, despite the absence of any
grossly apparent blood washout, tissue water evaluations in one FIGP
animal were indicative of osmotic dehydration and thus of some
perfusion.
The mesenteric, pancreatic, splanchic, and other small abdominal
vessels were largely free of blood by the conclusion of perfusion.
However, blood-filled vessels were not uncommon, and examination
during perfusion of mesenteric vessels performed with an
ophthalmoscope at 20X magnification revealed stasis in many smaller
vessels, and irregularly shaped small clots or agglutinated masses of
red cells in most of the mesenteric vessels. Nevertheless, despite
the presence of massive intravascular clotting, perfusion was
possible, and significant amounts of tissue water appear to have been
exchanged for glycerol.
One immediately apparent difference between the FGP and FIGP
animals was the accumulation in the lumen of the ileum of large
amounts of perfusate or perfusate ultrafiltrate by the ischemic
animals. Within approximately 10 minutes of the start of reperfusion,
the ileum of the ischemic animals that had been laparotomized was
noticed to be accumulating fluid. By the end of perfusion, the
stomach and the small and large bowel had become massively distended
with perfusate. Figure 14 shows both FIGP and FGP ileum at the
conclusion of glycerol perfusion. As can be clearly seen, the FIGP
intestine is markedly distended. Gross examination of the gut wall
was indicative of tissue-wall edema as well as intraluminal
accumulation of fluid. Often by the end of perfusion, the gut had
become so edematous and distended with perfusate that it was
impossible to completely close the laparotomy incision. Similarly,
gross examination of gastric mucosa revealed severe erosion with the
mucosa being very friable and frankly hemorrhagic.
Escape of perfusate/stomach contents from the mouth (purging)
which occurs during perfusion in ischemically injured human suspension
patients did not occur, perhaps due to greater post-mortem competence
of the gastroesophageal valve in the cat.
Oxygen consumption in the two ischemic cats in which it was
measured was dramatically impacted, being only 30% to 50% of control
and deteriorating throughout the course of perfusion (Figure 12).
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